The excessive use of fossil fuels accelerated global warming; however, most of the energy is still dependent on fossil fuels [1], and the acceleration of global warming caused by carbon dioxide will produce more environmental issues [2], [3], [4]. As an alternative to fossil fuels, researchers have sought alternative energy sources that are eco-friendly, renewable, and clean [5], [6], [7], [8]. Hydrogen energy could be an affordable alternative that does not emit greenhouse gases when burned, is eco-friendly, clean, reusable, and has a high energy density [9], [10], [11], [12]. To produce hydrogen traditionally, electrolytic, thermochemical, or biological routes can be used [13], [14]. Among the various hydrogen production methods, electrolytic and thermochemical methods require a large amount of energy to produce hydrogen, which has disadvantages in energy efficiency [15]. Biological hydrogen production has some strengths because it has a relatively low energy requirement, is a simple production method, and poses little threat to the environment [16], [17]. Hydrogen production processes are categorized based on their metabolic pathways: 1) biophotolysis using light energy and water by algae and cyanobacteria, 2) photofermentation using light energy and organic materials by photosynthetic microorganisms, and 3) dark fermentation using organic materials by fermentative bacteria [18]. Although several methods can produce hydrogen, dark fermentation has unique advantages over alternative methods, such as a lower energy requirement, and the ability to produce hydrogen from a wide range of substrates, including organic wastes [19], [20], which makes it an attractive option for sustainable hydrogen production.

Clostridium species are mainly used in dark fermentation methods for hydrogen production and have a mechanism for producing hydrogen through the reducing power of hydrogenases [21]. They are obligate anaerobic bacteria with higher hydrogen production yields than facultative anaerobic bacteria. Clostridium acetobutylicum has been studied as a producer of organic alcohols such as acetone, butanol, and ethanol. Furthermore, it is one of the Clostridium species with well-defined metabolic pathways [22], [23]. In the case of hydrogen in C. acetobutylicum, the carbon flux passes through the process, and hydrogen is produced through the pyruvate: ferredoxin oxidoreductase (PFOR) pathway [24]. Metabolic pathways from pyruvate also produce various organic acids and alcohols [25]. As a result, understanding the overall metabolic pathways and genetic modifications is essential for efficient hydrogen production [26], [27] and there are three possible methods to increase hydrogen production.

The gapC gene encodes for the glyceraldehyde-3-phosphate dehydrogenase (GAPDH), which generates NAD(P)H during the oxidation of glyceraldehyde-3-phosphate in glycolysis and can be used to increase the amount of NAD(P)H produced through the primary pathway, producing metabolites and energy [28], [29]. Through overexpression of the gapC gene, it was expected that the activation of the glycolysis system and the NADPH pool required for biochemical production would increase. The production of amino acids and biochemicals was examined in Corynebacterium, Escherichia coli, Vibrio, and Yarrowia [30], [31], [32], [33], [34], [35], [36], [37], suggesting that the overexpression of the gapC gene could be a reasonable target for increasing biochemical production. In addition, gapC from C. acetobutylicum shows a high preference for NADP to supply more NADPH, resulting in a favorable target to replace the original gapC in various strains [31], [37], [38]. Interestingly, there have been no reports on applying gapC to gaseous products such as hydrogen in C. acetobutylicum.

In recent years, there has been increasing interest in hydrogen production using biomass, with a significant number of studies being conducted in this area [39], [40]. Biomass pretreatment generates inhibitors such as Furfural and 5-hydroxymethylfurfural (HMF), which can hinder microbial growth and disrupt the subsequent fermentation process [41]. Aldehyde dehydrogenase requires NADH as a cofactor to decompose aldehydes present in biomass inhibitors [42]. On the other hand, GAPDH can produce NAD(P)H through glycolysis, and the use of this NAD(P)H as a cofactor is expected to enhance the activity of aldehyde reductase, thereby preventing inhibition caused by inhibitors.

In the present study, C. acetobutylicum ATCC 824 strain was selected and engineered to produce more hydrogen by overexpressing gapC. The overexpression of gapC affected hydrogen production, promoted carbon source consumption, and increased the NADPH/NADP+ pool. Additionally, an increase in the resistance to toxic byproducts was observed. Our results demonstrated the feasibility of overexpressing gapC for enhancing hydrogen production in C. acetobutylicum, offering new opportunities to utilize lignocellulosic biomass containing various carbon sources and inhibitors.